We are continuing our investigation of a series of temperature-sensitive mutants of Escherichia coli which carry mutations clustered in the aroE-rpsL (72 min) region of the bacterial chromosome. We have examined 21 conditional mutants from our collection. All of these strains show a reduction in growth rate at 44 C and most are killed by continued incubation at this temperature. Each also showed a reduced rate of protein synthesis at this restrictive temperature, measured as the rate of incorporation of radioactive amino acids into protein. Protein synthesis in vitro was also temperature-sensitive, when ribosomes from these mutants were used in protein synthesis assays. Alterations in 3 different small subunit proteins and 4 large subunit proteins have been detected in 8 different mutants by the method of two-dimensional polyacrylamide gel electrophoresis. Defects in ribosome assembly at the restrictive temperature and alterations in the sensitivity or resistance to 3 ribosomal antibiotics have been observed in a number of these strains. We are presently mapping the temperature-sensitive mutation in each strain and are conducting a variety of in vitro assays to better define the site and nature of the defective ribosomal activity in protein synthesis. We are also continuing our examination of the F' partial diploid strains we have which carry episomal DNA containing the ribosomal protein genes in the 72 min region of the chromosome of E. coli. We have examined the physical characteristics of the episomal DNA by an analysis of restriction endonuclease digests of F ion and F' DNA. We are presently examining the in vitro transcription products of these DNAs and are also continuing our studies on the types of in vitro translation products which can be identified in a DNA-dependent protein synthesizing system.